Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Although routine endoscopic screening and early surgical intervention has reduced the incidence of CRC mortality, the five-year survival rate of patients with advanced disease is less than 10%. Genomic studies have suggested key genes that drive CRC initiation and progression but this information has yet to produce effective new therapies, in part, because current models to validate cancer drivers and new therapeutic targets in CRC are slow and cumbersome. This proposal aims to develop and utilize new murine models of colorectal cancer (CRC) to define genetic requirements for cancer progression and therapy response. To this end, we implement a novel approach to validate cancer drivers and therapeutic targets using orthotopic colon stem cell transplants that form focal colon tumors and progress to malignant disease. This circumvents the problems associated with transgenic mouse models, which i) rarely develop colon tumors and ii) do not live long enough to develop metastases due to small intestinal tumor burden. We have previously developed a robust model of APC loss in the colon using short hairpin RNAs targeting APC and showed that APC loss is required for disease maintenance of the primary tumor (14). My plan focuses on two future directions from that study: i) to test if Apc restoration in later CRC stages (Stage II, III, or IV will result in disease regression, and ii) to test if Smad4 mutations abrogate the dependence on hyperactive WNT signaling conferred by Apc loss in CRC. Successful completion of the project will produce new CRC models, validate potential cancer drivers, and identify key determinants that influence the action of WNT pathway inhibitors.